The overall goal of this surgical technique is to standardize the analysis of bone healing in mice. This surgical technique can help answer key questions in bone healing research about the roots of bone morphogenetic proteins and muscular grow factors and the mechanisms of bone repair. The main advantages of this technique are the reproducibility of the femur osteotomy and a high axial and rotationist ability facilitated by the use of the intramedullary locking nail.
Demonstrating the procedure will be Tobias Fritz, a colleague of our department. One day before the surgery until three days after the surgery, add tramadol hydrochloride to the animal's drinking water for analgesia. On the day of the surgery, confirm the appropriate level of sedation of a 25 to 35 gram mouse, by a lack of response to toe pinch and place the animal under a heat radiator.
Shave the entire right hind leg of the animal and apply depilatory cream. After five minutes, remove the cream and clean the leg with water. Then disinfect the surgical site with 96%ethanol.
Under aseptic conditions, place the mouse in the supine position on the operating table with the right knee bent to allow an anterior approach of the knee condyles. Next, use a sterilized size 15 scalpel blade to make a five millimeter medial parapatellar incision in the right knee. Then use fine forceps to lift the exposed patellar ligament and use the blade to carefully mobilize the ligament.
Shifting the patella laterally with the blade to expose the intercondylar notch of the femur, use a hand drill equipped with a one millimeter centering bit, and held at a 45 degree offset to the femur axis to bore the intercondylar notch. Slowly changing the direction of the bit, drill until the bit parallels the bone axis of the femur, stopping when the intramedullary cavity is reached. When the intercondylar notch is open, insert a 24 gauge needle into the cavity over the entire length of the femur and rotate the needle to manually ring the intramedullary cavity.
Then replace the 24 gauge needle with a 27 gauge needle and push the 27 gauge needle forward to perforate the cortical bone of the femur proximally at the greater trochanter. Remove the 27 gauge needle and use the hand drill to implant the intramedullary nail through the intercondylar notch under continuous rotation and axial pressure until the distal end of the nail reaches the condyles. Now place the mouse in the left lateral position and use the scalpel to make a longitudinal skin incision along the diaphyseal region of the lateral femur from the hip joint to the knee joint to surgically expose the midshaft of the femur.
Using small preparation scissors, split the fascia and spread the muscles toward the femur axis from the lateral side until the diaphyseal region of the femur is exposed. Undermine the bone with the dressing forceps. Then spread the dressing forceps and retract the muscles to expose the femur.
Mount the aiming device to the distal end of the nail, advancing the device until it attaches to the adapter flange of the nail. Then turn the aiming device to an anterolateral angle to the femur, and use the centering one millimeter diameter drill bit to create a small cavity in the facing cortical bone at the proximal interlocking hole position. Next, with a 0.3 millimeter diameter drill bit, use the aiming device to drill a hole through both the facing and averted cortical bones.
Insert the proximal interlocking pin through the aiming device. The interlocking pin driveshaft will shear off as soon as the interlocking torque is achieved. When both the proximal and distal interlocking pins have been placed, equip the aiming device with the saw guide on the lateral side, between the two interlocking pins, and saw the bone with a Gigli saw under continuous irrigation with saline.
When the osteotomy is complete, cut the saw at end close to the bone and carefully remove the blade to avoid causing damage to the soft tissue. Remove the aiming device and use small pincers to clip off the remaining shaft of the intramedullary nail at the marked line. Then close the muscle layers at the lateral site of the femur, and use single sutures to close the skin.
At the anterior site of the knee, reposition the patella and fix the patella tendon to the muscles with one single suture, followed by closure of the wound with single sutures. Finally, allow the animal to recover under the heat lamp with monitoring, until fully recovered. The most significant complication that can occur is the incorrect implantation of the locking nail with the protrusion of the nail level to the condyles of the knee joint.
This mainly occurs due to the incorrect handling of the aiming device or the use of an animal with a too small femur, particularly in mice with body weights below 20 grams. Another complication is the dislocation of an interlocking pin, which is mainly caused by an incomplete insertion of the pin, and can be avoided by radiographic confirmation of the correct implant placement during, or immediately after, the surgery. Radiological analysis after five weeks confirms a complete healing of the 0.25 millimeter osteotomy gap, at which point, the periosteal callus is almost completely remodeled.
In contrast, in femora stabilized with a 2 millimeter gap, the osteotomy does not heal, and reliably exhibits an atrophic nonunion formation for up to 10 weeks of bone healing. After stabilization with a 0.25 millimeter osteotomy gap, histological analysis reveals a typical pattern of secondary fracture healing with callus formation, including intramembranous and endochondral ossification that is remodeled into lamellar bone at five weeks. In contrast, femora stabilized with two millimeter osteotomy gaps demonstrate an atrophic nonunion after 10 weeks that is associated with a high amount of fibrous tissue within the osteotomy gap, and no evidence of bone healing or bridging.
Once mastered, this technique can be completed in 20 minutes if it is performed properly. After watching this video, you should have a good understanding of how to use this technique as the standardest model for fracture healing research.
